1. Field of the Invention:
This invention relates to a column structure of liquid chromatography used for separating and purifying materials and a method of high-performance packing of gels into this column.
2. Discussion of the Background:
In conventional liquid chromatographic devices, a column wherein slurry dispersed with gel is packed by liquid-flow is generally used for the column as the main component for separation and purification.
This method of packing the gels by liquid-flow is called a slurry packing method and is generally operated as shown in FIGS. 5(a) through 5(e). A reservoir 4 is connected to the upper end of a column body 1 having a lower flange 2 blocking the bottom thereof. A homogeneous gel slurry 5 of predetermined quantity and density is poured into the reservoir from above (see FIG. 5(a)). Then, an upper flange 3 is attached to the upper end of the reservoir 4. And a gel bed 6 is formed within the column body 1 by liquid-flow through the column body and reservoir and pressurization with a high pressure pump (see the top and bottom openings for liquid flow and for pressurization, shown only in FIG. 5(b)). After the liquid-flow and pressurization, supernatant liquid in the reservoir 4 is discharged by a pump and the like (see FIG. 5(c)), and the reservoir 4 is removed from the column body 1. The gel bed 6 heaps up over the top of the column body 1 when the reservoir 4 is removed therefrom (see FIG. 5(d)).
After the heap of the bed is removed, the upper flange 3 is fixed to the top of the column body 1 to finish the packing operation (see FIG. 5(e)).
This slurry packing method has been practiced as one of most common and superior packing methods because the advantages that stable gel beds and remarkably increased performance of the column can be obtained.
However, in conjunction with recent developments in biotechnology and its increasing importance to studies in related fields of biochemistry, many kinds of gels for liquid chromatography have been in development. It has been desired that the users can themselves easily pack and replace these gels into a column which is most suitable for the objective ingredients and matching size (diameter and length) for a required quantity to be treated.
In order to so form stable gel beds in a column for separation and purification or to fill, as another object, a void in the top of liquid inlet due to the volume change of the gel bed during the liquid chromatographic operation, a method to move a liquid dispersing plate and a liquid collecting plate situated at the upper and lower ends of the inside of the column has been proposed. A device utilizing this method to move a liquid dispersing plate (or a liquid collecting plate) may be classified as an axially adjustable-type column for preparative liquid chromatography interconnected with a pneumatic, hydraulic, or mechanical driving mechanism for compressing and pressurizing gel beds and the method may be called a pressurized packing method.
An example utilizing such a pressurized packing method as disclosed in Japanese Patent Publication No. Sho 58-20284 is shown in FIG. 6. In FIG. 6, a piston-like sliding body 8 having a head 9 is fit liquid-tightly and vertically movably in the column body 10. The liquid collecting plate 13 is attached to the upper end of the above mentioned head 9. While the sliding body is at the lower dead point of its travelling path, homogeneous gel slurry 11 is poured into the column body 10. After that, the liquid dispersing plate 16 and a cover 12 are fixed to a fixing flange 14 on top of the column body.
When the sliding body 8 is moved upward from its lower dead point by a drive shaft 15, for example by utilizing a pneumatic or hydraulic jack, pressure is applied to the homogeneous gel slurry 11 to form the gel bed. Removal of the gel bed for replacement is performed by lifting the sliding body 8 with the cover 12 removed. Then it is ready for the next chromatographic operation.
FIG. 7 shows another example of the pressurized packing method as disclosed in Japanese Laid-Open Patent Application No. Sho 61-28864. A column for liquid chromatography 23 is fixedly attached to a column pedestal 24. After the homogeneous gel slurry is poured through the top of the column 23, an adjustable plug (not shown) is attached to the top of the inside of the column. A shaft 22 connected with this adjustable plug is extended upward out of the column. The upper end thereof is fixed to an adjustable plug transfer pedestal 21. Under this condition, threaded rotation shafts 18 are rotated, via the reducing mechanism 19, by the rotation of a motor 20. Since rotation shafts 18 are threaded in the transfer pedestal 21, they cause the pedestal to move vertically. A gel bed is formed by lowering the adjustable plug transfer pedestal 21 and the adjustable plug at a fixed rate to apply pressure to the homogeneous gel slurry in the column 23 and by discharging only the liquid to the outside through piping on both sides of the column (not shown). When the gel in the column is no longer usable, the lower flange 17 is removed, the adjustable plug transfer pedestal 21 is lowered, and the gel is discharged from the bottom of the column 23 to prepare the column for the subsequent packing.
Though quite different from the above method which applies pressure for the formation of a gel bed, a column of such a structure that an adjustable plug is built therein has also been proposed for packing by pressure the void at the liquid inlet in the column, which void occurs due to the volume change of the gel bed during the liquid chromatographic operation. An example is disclosed in Japanese Utility Model Publication No. Sho 43-22959, and shown in FIG. 8. In the figure, if the top of the gel bed 25 is lowered in the course of time due to the inflow of liquid through a treating liquid inflow passage 29, pressurized gas is supplied into a pressurized gas chamber 27 through a flow-in duct 26 and an adjustable body 28 always applies pressure on the gel bed 25 and moves downward as required. Therefore, the void is not formed during that time. Element 30 is a seal ring for sealing air and liquid.
In an axially adjustable column for separation and purification according to the conventional pressurized packing method, a special mechanism is required to constantly maintain the travel speed of the adjustable plug by a pneumatic, hydraulic or mechanical driving mechanism and to provide a large overload for pressurizing the gel bed as in the cases of Japanese Patent Publication No. Sho 58-20284 or Japanese Laid-Open Patent Application No. Sho 61-28864, described above, in order to move either one or both of the liquid dispersing plate and the liquid collecting plate. In order to meet with these requirements, the pneumatic, hydraulic or mechanical driving mechanism will inevitably be structured in large scale.
Moreover, such a mechanical driving mechanism forms a gel bed by pressing it with a strong force, and accordingly the density distribution of the gel bed is liable to become uneven in the axial direction, and it is difficult to achieve a high column performance as has been attained by conventional slurry packing methods.
The method described in Japanese Utility Model Publication No. Sho 43-22959 has as its object to fill the void which arises between the gel bed and the adjustable plug during liquid chromatographic operation, and has no object to form the gel bed. Further this method seems to provide no effective method for forming the bed. For instance, even if this method of moving the adjustable plug with use of pressurized gases is applied to the gel bed formation, the transfer speed of the adjustable plug cannot be controlled at a fixed rate due to the cushion effect of the pressurized gas, and desired separation performance cannot be constantly accomplished. And when leakage happens to occur at the sealing surface between the adjustable plug and the inner wall of the column, a problem arises that separation performance of the column will be decreased drastically due to the flow of the pressurized gases through the gel bed.
As has been described above, in contrast to the slurry packing method wherein high column performance is achieved, it is difficult to form a satisfactory gel bed by any of the conventional pressure packing methods described in Japanese Patent Publication No. Sho 58-20284 and Japanese Laid-Open Patent Application No. Sho 61-28864, or the method of Japanese Utility Model Publication No. Sho 43-22959 utilizing the similar axially adjustable-type column.
It may be said that the features of the conventional slurry packing method for obtaining high performance and those of the pressurized packing method for rapidly forming the gel bed effectively by applying pressure on the adjustable plug are mutually exclusive.